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An accumulating number of studies suggest the potential of circulating cell-free microbial DNA (cfmDNA) as a non-invasive biomarker in various diseases, including cancers. However, its value in the prediction or prognosis of clinical outcomes of immune checkpoint inhibitors (ICIs) is poorly explored. The circulating cfmDNA pool may also reflect the translocation of various microbial ligands to the circulatory system and may be associated with the increased release of soluble CD14 (sCD14) by myeloid cells. In the present study, blood samples were collected from advanced melanoma patients (n = 66) before and during the anti-PD-1 therapy (approximately 3 and 12 months after the start). Then, V3-V4 16S rRNA gene sequencing was performed to analyze the circulating cfmDNA extracted from plasma samples. Moreover, the concentration of plasma sCD14 was measured using ELISA. As a result, the differences in the circulating cfmDNA profiles were found between patients with favorable and unfavorable clinical outcomes of the anti-PD-1 and baseline signatures correlated with progression-free survival and overall survival. Moreover, there was a higher concentration of plasma sCD14 in patients with unfavorable clinical outcomes. High baseline sCD14 level and its increase during the therapy prognosticated worse survival outcomes. Taken together, this preliminary study indicates the potential of circulating cfmDNA signatures and plasma sCD14 levels as biomarkers of clinical outcomes of ICIs. Full article

Long COVID, also called post-acute sequelae of SARS-CoV-2, is characterized by a multitude of lingering symptoms, including impaired cognition, that can last for many months. This symptom, often called “brain fog”, affects the life quality of numerous individuals, increasing medical complications as well as healthcare expenditures. The etiopathogenesis of SARS-CoV-2-induced cognitive deficit is unclear, but the most likely cause is chronic inflammation maintained by a viral remnant thriving in select body reservoirs. These viral sanctuaries are likely comprised of fused, senescent cells, including microglia and astrocytes, that the pathogen can convert into neurotoxic phenotypes. Moreover, as the enteric nervous system contains neurons and glia, the virus likely lingers in the gastrointestinal tract as well, accounting for the intestinal symptoms of long COVID. Fusogens are proteins that can overcome the repulsive forces between cell membranes, allowing the virus to coalesce with host cells and enter the cytoplasm. In the intracellular compartment, the pathogen hijacks the actin cytoskeleton, fusing host cells with each other and engendering pathological syncytia. Cell–cell fusion enables the virus to infect the healthy neighboring cells. We surmise that syncytia formation drives cognitive impairment by facilitating the “seeding” of hyperphosphorylated Tau, documented in COVID-19. In our previous work, we hypothesized that the SARS-CoV-2 virus induces premature endothelial senescence, increasing the permeability of the intestinal and blood–brain barrier. This enables the migration of gastrointestinal tract microbes and/or their components into the host circulation, eventually reaching the brain where they may induce cognitive dysfunction. For example, translocated lipopolysaccharides or microbial DNA can induce Tau hyperphosphorylation, likely accounting for memory problems. In this perspective article, we examine the pathogenetic mechanisms and potential biomarkers of long COVID, including microbial cell-free DNA, interleukin 22, and phosphorylated Tau, as well as the beneficial effect of transcutaneous vagal nerve stimulation. Full article

Human blood contains low biomass of circulating microbial cell-free DNA (cfmDNA) that predominantly originates from bacteria. Numerous studies have detected circulating cfmDNA in patients with infectious and non-infectious diseases, and in healthy individuals. Remarkable differences were found in the microbial composition of healthy subjects and patients compared to cohorts with various diseases or even patients with diversified prognoses, implying that these alterations may be associated with disease development. Although the function of circulating cfmDNA needs to be elucidated (whether it acts as a bystander of dysbiosis or a key player in disease development), several studies have demonstrated its potential as a non-invasive biomarker that may improve diagnosis and treatment efficacy. The origin of circulating cfmDNA is still the subject of much deliberation, but studies have identified members of various microbiome niches, including the gut, oral cavity, airways, and skin. Further studies investigating the origin and function of circulating cfmDNA are needed. Moreover, low-biomass microbiome studies are prone to contamination, therefore stringent negative experimental control reactions and decontamination frameworks are advised in order to detect genuine circulating cfmDNA. Full article

Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals. Full article